Modulation of 4-AP block of a mammalian A-type K channel clone by channel gating and membrane voltage.
Modulation of 4-AP block of a mammalian A-type K channel clone by channel gating and membrane voltage.
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DOI：
10.1016/s0006-3495(94)80462-x
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发表时间：
1994-07
影响因子：
3.4
通讯作者：
Jian-An Yao;G. Tseng
中科院分区：
文献类型：
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作者：
Jian-An Yao;G. Tseng
我们研究了4-AP和哺乳动物A型K通道克隆（rKv1.4）表达在非洲爪蟾卵母细胞使用全细胞和单通道记录之间的相互作用的状态，电压和时间依赖性。4-AP从细胞膜的胞质侧阻断rKv1.4。街区的发展需要渠道的开放。通过去除通道的快速失活门（称为“Del A”的缺失突变体）来加强阻断。一个短脉冲序列，激活rKv1.4没有失活诱导更多的阻滞4-AP比一个长脉冲，激活，然后失活的通道。这些观察结果表明，激活和失活门都限制了4-AP与通道的结合。在通道开放期间也发生4-AP的解除阻断，因为解除阻断需要去极化，并且通过更频繁或更长的去极化脉冲加速（使用依赖性解除阻断）。对阻断发展速率的浓度依赖性分析表明，4-AP以缓慢的动力学阻断rKv1.4（在-20 mV下，结合和解结合速率常数分别为3.2 mM-1 s-1和4.3 s-1）。这与单通道记录一致：4-AP在爆发内的快速开放和关闭动力学中引起很少或没有变化，但缩短了平均爆发持续时间，更重要的是，降低了去极化通道开放的概率。去极化可能降低开放通道中4-AP结合位点的亲和力，因为更强的去极化降低了4-AP的稳态阻断程度。此外，在去极化保持电压下建立4-AP阻滞后，进一步去极化诱导时间依赖性解除阻滞。我们的数据表明，4-AP结合到开放的rKv1.4通道，并从开放的rKv1.4通道中解结合，动力学缓慢，结合位点的可及性由通道门控装置控制，结合位点的亲和力由膜电压调节。
We examined the state-, voltage-, and time dependences of interaction between 4-AP and a mammalian A-type K channel clone (rKv1.4) expressed in Xenopus oocytes using whole-cell and single-channel recordings. 4-AP blocked rKv1.4 from the cytoplasmic side of the membrane. The development of block required channel opening. Block was potentiated by removing the fast inactivation gate of the channel (deletion mutant termed "Del A"). A short-pulse train that activated rKv1.4 without inactivation induced more block by 4-AP than a long pulse that activated and then inactivated the channel. These observations suggest that both activation and inactivation gates limit the binding of 4-AP to the channel. Unblock of 4-AP also occurred during channel opening, because unblock required depolarization and was accelerated by more frequent or longer depolarization pulses (use-dependent unblock). Analysis of the concentration dependence of rate of block development indicated that 4-AP blocked rKv1.4 with slow kinetics (at -20 mV, binding and unbinding rate constants were 3.2 mM-1 s-1 and 4.3 s-1). This was consistent with single-channel recordings: 4-AP induced little or no changes in the fast kinetics of opening and closing within bursts, but shortened the mean burst duration and, more importantly, reduced the probability of channel opening by depolarization. Depolarization might decrease the affinity of 4-AP binding site in the open channel, because stronger depolarization reduced the degree of steady-state block by 4-AP. Furthermore, after 4-AP block had been established at a depolarized holding voltage, further depolarization induced a time-dependent unblock. Our data suggest that 4-AP binds to and unbinds from open rKv1.4 channels with slow kinetics, with the binding site accessibility controlled by the channel gating apparatus and binding site affinity modulated by membrane voltage.